Citrus hystrix essential oil (CHEO) have shown various pharmacological properties including antibacterial activity. This EO also possessed antibacterial effect against foodborne pathogens. There is less information available about the synergy interaction between CHEO and tetracycline, as well as their mechanism of action. Therefore, this study was conducted to evaluate the synergistic effect of CHEO and tetracycline against clinical isolate of Escherichia coli. Antibiofilm, bacteriolytic, and efflux pump inhibitor activities were also performed. The chemical composition of CHEO was analysed using GC-MS. Three major compounds, D-limonene (25.02%), β-pinene (23.37%), and β-sabinene (22.20%) were identified. CHEO exhibited moderate antibacterial activity with MIC value of 250 μg/mL. The combination of CHEO (7.8 μg/mL) and tetracycline (62.5 μg/mL) produced a synergistic effect on E. coli with fractional inhibitory concentration index of 0.5. This mixture inhibited biofilm formation in E. coli. The combination of 7.8 μg/mL CHEO and 62.5 μg/mL tetracycline demonstrated bacteriolytic activity. In addition, the CHEO at 250 μg/mL showed a significant effect in inhibiting efflux pump. D-limonene has a binding free energy value of -20.13 kcal/mol with ompA transmembrane domain of E. coli. This finding indicates that CHEO has a potency to be developed as natural antibacterial against E. coli.